Bacteria can form multi-cellular communities in which individual cells are protected from environmental insults such as antibiotics by virtue of being (1 encased in a protective matrix comprised of polysaccharides and other macromolecules and (2) physiologically distinct from free-living, planktonic cells. Furthermore, biofilm formation enhances the ability of bacteria to colonize surfaces, including host tissues and abiotic surfaces such as medical implants. As a result of these characteristics, bacteria in biofilms are responsible for the majority of hospital-acquired infections. Due to their medical relevance, how biofilms form and disperse from such biofilms is being intensively studied. Although numerous animal models of biofilm formation have been developed, few robustly demonstrate that mechanisms of biofilm formation uncovered in culture reflect what actually occurs in nature. One such robust model, however, can be found in the Vibrio fischeri - squid (Euprymna scolopes) symbiosis. To colonize, V. fischeri first forms a biofilm on the surface of the symbiotic organ, then disperses from it to enter and ultimately colonize sites deep within this organ. Our work has shown that genes required for biofilm formation in laboratory culture are similarly required for host-associated biofilm formation and colonization. Furthermore, genetic conditions that enhance biofilm formation in laboratory culture also strikingly enhance host-associated biofilm formation and colonization. This strong correlation affords us an exceptional opportunity to develop and test hypotheses about the mechanisms of biofilm formation and dispersal in bacterial colonization of a eukaryotic host. Our work has revealed that biofilm formation and colonization depends on syp, an 18-gene locus involved in the production and export of a polysaccharide, and on regulators that control syp transcription. More recently, we determined that V. fischeri exerts considerable post-transcriptional control over the activities of specific Sp proteins, including SypA, a protein that is controlled by phosphorylation on a serine residue. SypA must be unphosphorylated to promote biofilm formation and colonization, but how this small single domain protein functions remains unknown and will be investigated here. We also propose to delve more deeply into the dynamics of host- associated biofilm formation and the factors that contribute to those dynamics and the ability of V. fischeri to become the dominant microbe within host-associated biofilms. Finally, we will probe the dispersal process, an event that clearly occurs in the context of symbiotic colonization by V. fischeri, but for which little i currently known. These directions, a mix of both mechanistic characterization of known factors and the exploration of unknown pathways, have as their central focus an understanding of the processes that are occurring naturally in host-associated bacteria. We anticipate that this work will provide insights into the mechanisms by which bacteria respond to their environment and transition in and out of multi-cellular communities within an animal host.